There are many different laboratory techniques required to manufacture proteins. These techniques can be broken up into three groups: cell culture, protein purification, and protein modification.
Cell culture processes work by growing cells in vitro (outside of the body) with nutrients that allow them to grow and divide. Because biological products like proteins depend on cells’ ability to reproduce, cells are often grown through culturing.
Culturing cells requires that they be placed in a system of nutrients and then controlled for temperature, oxygen content, pH level, etc. In this article, we will look at different laboratory techniques of protein expression.
Pichia pastoris (Yeast)
When it comes to manufacturing proteins, there are a large number of different expression systems to choose from. However, we’ll discuss the pichia pastoris expression system here, which is one of the most common and proficient systems.
The pichia pastoris (Yeast) system is a eukaryotic expression vector that allows for the large-scale production of recombinant proteins. Ribosomes are the cellular organelle where protein synthesis takes place. It’s here that tRNA (a molecule involved in protein synthesis) enters the A-site of ribosomes, and protein synthesis then takes place.
In order for this to occur, mRNA (which is a molecule that codes for specific amino acids in a certain sequence) attaches itself to the small ribosomal subunit. The entire process occurs in both prokaryotic and eukaryotic organisms.
Escherichia coli (E. coli)
E.coli is a bacterial species commonly found in the human intestine. However, this bacteria also has many applications in the field of protein manufacturing and is often used in laboratories for this specific purpose.
E. coli manufactures proteins through its natural life cycle, but this process can be mimicked in a laboratory setting by the addition of calcium to a bacterial culture. The presence of calcium causes spontaneous protein secretion through a process known as bacterial co-expression.
In this method, genes from an origin of interest are inserted into E. coli and forced into its natural life cycle where it will begin producing proteins. In order to increase the yield of proteins, proteins are often tagged with an affinity tag, which enables their purification through chromatography or precipitation.
The purification process can be further optimized by removing any bacterial or E. coli based contaminants from the final product. Given that E. coli is a highly prevalent bacteria in many laboratories, it presents a high risk to contaminate a protein sample with E. coli based impurities if adequate precautions are not taken.
Proteins from E. coli typically have 70% homology compared to natural proteins. This decreases the chance that proteins produced by E. coli can be mistaken for endogenous genes and associated with autoimmune disorders in humans.
Trichoderma reesei is a type of filamentous fungus that when grown in culture produces proteins with human-like amino acid sequences. This makes it an ideal candidate to produce recombinant protein for pharmaceutical applications because it has been shown to have the ability to process ‘difficult’ substrates such as hydrophobic peptides, or proline-rich peptides up to 100 residues long.
However, the difference between Trichoderma reesei and other filamentous fungi that are used for protein production is that it can be grown in submerged fermentation conditions – similar to those typically used for E. coli strains which enables the use of recombinant DNA techniques (typically E. coli strains are grown on agar plates).
This makes Trichoderma reesei more amenable to manipulation than other filamentous fungi. Trichoderma reesei has been used as a protein-expression system since the 1980s, but it was first widely exploited in 1998 by the Danish company Novo Nordisk to produce recombinant human insulin on a large scale and at low cost – this was an important milestone as it was the first time a recombinant human protein had been manufactured on an industrial scale.
Saccharomyces cerevisiae expression system is the process of manufacturing proteins through expression in yeast cells. This technique can be used to produce a wide variety of proteins for clinical applications.
Proteins are manufactured by first transcribing DNA into RNA, known as transcription. The RNA sequence is then read by the ribosome, which translates it into an amino acid sequence, known as translation. The amino acid sequence is then folded into a three-dimensional structure, known as the tertiary structure. Folding must take place for proper function.
The DNA sequence to be translated in the S cerevisiae expression system is usually isolated from the cells of mammals or other sources, purified, and inserted into yeast vectors with promoter sequences attached.
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Bacillus subtilis is a Gram-positive bacterium used for commercial protein expression. Typically, commercial proteins are manufactured by bacteria. Proteins are made up of amino acids strung together in sequence according to the genetic code of DNA present in the gene sequence.
Each cell is capable of converting nucleic acid into protein, thus the process of producing proteins is often referred to as “expression.” The most common expression systems used today are bacteria and yeast.
Bacillus subtilis is an autotrophic bacterium that grows by producing amylase, protease, and phytase. These enzymes are then used to break down starch, protein, and phytic acid (an inhibitor of dietary mineral absorption in humans) respectively. As the bacteria grow, the number of copies of each gene increases within the cell.
Bacillus subtilis requires a nutrient medium with a carbon source, nitrogen source, and inorganic salts to grow, as well as temperature ranging from 20-30 degrees Celsius for optimal growth rate. Additionally, B.subtilis is capable of growing both aerobically and anaerobically.
When B. Subtilis is grown aerobically, only one plasmid copy is required for high-level protein expression. This makes it easy to maintain a recombinant strain in a production environment by minimizing the possibility of plasmid loss. A feature that makes B. subtilis protein expression system highly desirable for commercial use.
Protein manufacturing through recombinant DNA technology is an efficient method of producing high quantities of purified, non-endogenous proteins in the necessary quantities required for further research or clinical trials. The above are the most commonly used expression systems for commercial protein production. With this knowledge, it is possible to choose a protein expression system that is best suited for the application.